110. Performance of Different AAV Serotype Vectors Following Injection into the Deep Cerebellar Nuclei of ASMKO Mouse Brain

Abstract

Niemann-Pick A disease (NPA) is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Consequent accumulation of sphingomyelin and other lipids in the CNS results in the development of a rapidly progressive neurodegenerative disease with death occurring by 2 to 3 years of age. Previously we have shown that the storage pathology in the ASM knockout (ASMKO) mouse brain is amenable to AAV2/2-mediated gene therapy. Interestingly, correction of storage pathology occurred not only at the injection site, but also in regions that send and/or receive input from the injection site|[ndash]|suggesting that AAV vector and/or expressed ASM protein underwent transport. The present experiment evaluated the relative ability of recombinant AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8 serotype vectors encoding human ASM to facilitate gene transduction, express ASM protein, correct cholesterol storage pathology, undergo transport, rescue Purkinje cells, and initiate functional recovery in the ASMKO mouse. Male ASMKO mice (|[sim]|7 weeks old) were unilaterally injected with the different AAV serotype vectors within the deep cerebellar nuclei of the cerebellum (DCN). The DCN was targeted because it is highly connected with the CNS; and therefore, may provide a means to achieve widespread ASM expression throughout the brain. Mice were sacrificed at 14 weeks of age after undergoing rotarod testing. Mice injected with AAV2/1 and AAV2/8 demonstrated significant functional improvement on the rotarod, whereas mice injected with AAV2/2, AAV2/5 and AAV2/7 did not. Consistent with the behavioral results, cerebellar ASM protein levels (as detected by ELISA) were significantly higher in mice injected with AAV2/1 and AAV2/8, than mice injected with AAV2/2, AAV2/5, AAV2/7 and control mice. Preservation of Purkinje cells based on calbindin immunostaining was greatest in mice injected with AAV2/1 and AAV2/8. In all AAV-ASM treated mice, expression of ASM led to widespread clearance of filipin/cholesterol staining in the cerebellum, brainstem, and midbrain|[ndash]|indicating that AAV vector and/or expressed ASM underwent transport from the DCN. Overall, a positive relationship between ASM protein levels, filipin clearance, Purkinje cell survival, and rotarod performance was observed. These results support the further evaluation of AAV2/1 and AAV2/8-based vectors for gene therapy of the CNS manifestations in Niemann-Pick A disease.